PROJECT SUMMARY Liver receptor homolog-1 (LRH-1) is a nuclear receptor (NR) that has shown promise as an anti-diabetic therapeutic in murine studies and regulates cholesterol transport, bile acid biosynthesis, steroidogenesis, and glucose homeostasis, making it an attractive target for treating a variety of diseases. NRs are allosteric effectors, transmitting ligand binding status to alter target gene expression by selectively recruiting coregulator enzymes that modify chromatin and recruit transcriptional machinery. Although phospholipids are endogenous ligands of LRH-1, my lab has used structure-activity relationship (SAR) studies to develop synthetic agonists that are more useful for targeting this protein in experimental and clinical contexts. We have used a structure-guided approach to directly contact residues in the binding pocket to produce compounds that bind and activate LRH-1 with low- nanomolar potency. However, our understanding of how these small molecules drive LRH-1 activation through altered coregulator preference remains limited, and work on our current series of agonists has enhanced affinity of small molecules while minimally improving in-cell fold activation of LRH-1. How LRH-1 senses ligand to recruit coregulators is poorly understood, as evidenced by our high-affinity small molecules that do not efficiently drive coregulator association to enhance activation. Therefore, I will examine how coregulator preference is influenced by synthetic agonist binding and make modifications to small molecules that will directly target the LRH-1 activation function surface (AFS), the coregulator binding interface. I hypothesize that synthetic agonists induce conformational changes favoring interaction with coactivators and that LRH-1 activity can be modulated by directly altering AFS dynamics. In Aim 1, I will examine the mechanism of ligand-mediated activation by determining how coregulator recruitment is influenced by agonist binding. I will use fluorescence polarization (FP) and bioluminescence resonance energy transfer (BRET) binding assays to test how small molecules and endogenous phospholipids alter coregulator preference. I will then establish functional relevance for observed interactions using a luciferase reporter assay to examine whether ligand-mediated activation of LRH-1 relies upon expression of these coregulators. In Aim 2, I will use a structure-guided approach to make modifications that directly modulate AFS conformational dynamics. Using FP competition and luciferase reporter assays, I will identify how modifications impact binding and in-cell activation of LRH-1. Promising small molecules will be studied further with both X-ray crystallography and hydrogen deuterium exchange coupled with mass spectrometry (HDX-MS) to examine how these modifications drive AFS conformation and dynamics. The long-term goal of my work is to enhance design of small molecule modulators of LRH-1 activity that will be useful for probing LRH-1 biology and treating metabolic diseases.